PROJECT SUMMARY The envelope of Gram-negative bacteria is delimited by two lipid bilayers, the inner and outer membranes (IM and OM, respectively). The external leaflet of the OM contains densely packed lipopolysaccharides (LPS) that confer unusually high impermeability towards small hydrophobic molecules. As a result, Gram-negative bacteria are naturally resistant to many antibiotics. The IM and OM are separated by the aqueous compartment known as the periplasm where a cell wall composed of peptidoglycan resides. The peptidoglycan cell wall is an essential polymeric rigid structure that protects cells from osmotic lysis. Given the structural and protective functions of the cell envelope, proper envelope biogenesis is crucial for the survival of bacteria in many environments. Underscoring this is the fact that many antibiotics target envelope biogenesis pathways. Our long-term goal is to understand at the molecular level how Gram-negative bacteria build their cell envelope. Here, we propose to primarily use a combination of genetic and biochemical approaches to investigate two highly conserved systems that transport glycolipids across the cell envelope from their site of synthesis to the cellular compartment where they function: 1) MurJ, a polytopic IM protein that facilitates the most poorly understood step in peptidoglycan biosynthesis, the translocation of the lipid-linked peptidoglycan precursor lipid II across the IM; and 2) Lpt (LPS transport), a multi-protein bridge that spans the envelope and that functions to transport LPS from the IM to the cell surface. Both of these systems are essential for the viability of many bacteria including our model organism Escherichia coli. In aim 1, we propose studies to understand the mechanism that MurJ uses to flip lipid II by: a) conducting structure-function studies on MurJ; b) determining how MurJ interacts with lipid II; c) probing conformational changes that MurJ undergoes during the transport cycle; and, d) studying how MurJ is powered. In aim 2, we will investigate the most poorly understood step in LPS transport by focusing our studies on the LptFGB2C sub-complex, a unique ATP- binding cassette transporter that powers the extraction of LPS from the IM and its transport along the Lpt bridge to the cell surface. Specifically, in aim 2, we will: a) determine the topology of the membrane components LptF and LptG with respect to the IM; b) define protein-protein interactions in the LptFGB2C sub- complex; and c) elucidate how LptFGB2C couples ATP binding and hydrolysis in the cytoplasm to the extraction of LPS from the outer leaflet of the IM. Because inhibition of MurJ function leads to cell lysis and defects in the Lpt system can either increase OM permeability to many antibiotics or even cause death, knowledge gained from the proposed work will help in developing novel antimicrobial therapies. Studies on Lpt are especially needed to understand how we can overcome the innate resistance to antibiotics that Gram- negative have because of the barrier imposed by the presence of LPS at the cell surface.